Protein kinases are enzymes that catalyze the phosphorylation of hydroxyl groups of tyrosine, serine, and threonine residues of proteins. Many aspects of cell life (for example, cell growth, differentiation, proliferation, cell cycle and survival) depend on protein kinase activities. Furthermore, abnormal protein kinase activity has been related to a host of disorders such as cancer and inflammation. Therefore, considerable effort has been directed to identifying ways to modulate protein kinase activities. In particular, many attempts have been made to identify small molecules that act as protein kinase inhibitors.
The c-Met proto-oncogene encodes the Met receptor tyrosine kinase. The Met receptor is a 190 kDa glycosylated dimeric complex composed of a 50 kDa alpha chain disulfide-linked to a 145 kDa beta chain. The alpha chain is found extracellularly while the beta chain contains transmembrane and cytosolic domains. Met is synthesized as a precursor and is proteolytically cleaved to yield mature alpha and beta subunits. It displays structural similarities to semaphorins and plexins, a ligand-receptor family that is involved in cell-cell interaction. The ligand for Met is hepatocyte growth factor (HGF), a member of the scatter factor family and has some homology to plasminogen (Longati, P. et al., Curr. Drug Targets 2001, 2, 41-55); Trusolino, L. and Comoglio, P. Nature Rev. Cancer 2002, 2, 289-300].
Met functions in tumorigenesis and tumor metastasis. Expression of Met along with its ligand HGF is transforming, tumorigenic, and metastatic (Jeffers, M. et al., Oncogene 1996, 13, 853-856; Michieli, P. et al., Oncogene 1999, 18, 5221-5231). MET is overexpressed in a significant percentage of human cancers and is amplified during the transition between primary tumors and metastasis. Numerous studies have correlated the expression of c-MET and/or HGF/SF with the state of disease progression of different types of cancer (including lung, colon, breast, prostate, liver, pancreas, brain, kidney, ovaries, stomach, skin, and bone cancers). Furthermore, the overexpression of c-MET or HGF have been shown to correlate with poor prognosis and disease outcome in a number of major human cancers including lung, liver, gastric, and breast. c-MET has also been directly implicated in cancers without a successful treatment regimen such as pancreatic cancer, glioma, and hepatocellular carcinoma.
Met mutants exhibiting enhanced kinase activity have been identified in both hereditary and sporadic forms of papillary renal carcinoma (Schmidt, L. et al., Nat. Genet. 1997, 16, 68-73; Jeffers, M. et al., Proc. Nat. Acad. Sci. 1997, 94, 11445-11500). HGF/Met has been shown to inhibit anoikis, suspension-induced programmed cell death (apoptosis), in head and neck squamous cell carcinoma cells. Anoikis resistance or anchorage-independent survival is a hallmark of oncogenic transformation of epithelial cells (Zeng, Q. et al., J. Biol. Chem. 2002, 277, 25203-25208).
Increased expression of Met/HGF is seen in many metastatic tumors including colon (Fazekas, K. et al., Clin. Exp. Metastasis 2000, 18, 639-649), breast (Elliott, B. E. et al., 2002, Can. J. Physiol. Pharmacol. 80, 91-102), prostate (Knudsen, B. S. et al., Urology 2002, 60, 1113-1117), lung (Siegfried, J. M. et al., Ann. Thorac. Surg. 1998, 66, 1915-1918), and gastric (Amemiya, H. et al., Oncology 2002, 63, 286-296). HGF-Met signaling has also been associated with increased risk of atherosclerosis (Yamamoto, Y. et al., J. Hypertens. 2001, 19, 1975-1979; Morishita, R. et al., Endocr. J. 2002, 49, 273-284) and increased fibrosis of the lung (Crestani, B. et al., Lab. Invest. 2002, 82, 1015-1022).
Ax1 belongs to the subfamily of receptor tyrosine kinases (RTKs) that also includes Tyro3 and Mer. It was originally cloned from patients with chronic myelogenous leukemia and, when overexpressed, it exhibits transforming potential. Ax1 overexpression has been reported in a variety of human cancers, and is associated with invasiveness and metastasis in lung, prostate, breast, and gastric cancers as well as in renal cell carcinoma and glioblastoma. A recent study showed that Ax1 overexpression via a “tyrosine kinase switch” leads to resistance to imatinib in gastrointestinal stromal tumors. Ax1 expression is induced by chemotherapy drugs and overexpression of Ax1 confers drug resistance in acute myeloid leukemia. Ax1 has also been shown to regulate endothelial cell migration and tube formation. These findings suggest that Ax1 may be involved in the regulation of multiple aspects of tumorigenesis (for review, see Li et at, Oncogene 2009, 28: 3442).
Anaplastic lymphoma kinase (ALK) belongs to the receptor tyrosine kinase (RTK) superfamily of protein kinases. ALK expression in normal adult human tissues is restricted to endothelial cells, pericytes, and rare neural cells. Oncogenic, constitutively active ALK fusion proteins are expressed in anaplastic large cell lymphoma (ALCL) and inflammatory myofibroblastic tumors (IMT) due to t2; chromosomal translocations. ALK has also recently been implicated as an oncogene in a small fraction of non-small-cell lung cancers and neuroblastomas (Choi et al, Cancer Res 2008; 68: (13); Webb et al, Expert Rev. Anticancer Ther. 9(3), 331-356, 2009).
Anaplastic large-cell lymphomas (ALCLs) are a subtype of the high-grade non-Hodgkin's family of lymphomas with distinct morphology, immunophenotype, and prognosis. ALCLs are postulated to arise from T cells and, in rare cases, can also exhibit a B cell phenotype. In addition, there are 40% of cases for which the cell of origin remains unknown and that are classified as “null”. First described as a histological entity by Stein et al. based on the expression of CD30 (Ki-1), ALCL presents as a systemic disease afflicting skin, bone, soft tissues, and other organs, with or without the involvement of lymph nodes. ALCL can be subdivided into at least two subtypes, characterized by the presence or absence of chromosomal rearrangements between the anaplastic lymphoma kinase (ALK) gene locus and various fusion partners such as nucleophosmin (NPM). Approximately 50-60% of cases of ALCL are associated with the t(2;5;)(p23;q35) chromosomal translocation, which generates a hybrid gene consisting of the intracellular domain of the ALK tyrosine kinase receptor juxtaposed with NPM. The resulting fusion protein, NPM-ALK has constitutive tyrosine kinase activity and has been shown to transform various hematopoietic cell types in vitro and support tumor formation in vivo. Other less frequent ALK fusion partners, e.g., tropomyosin-3 and clathrin heavy chain, have also been identified in ALCL as well as in CD30-negative diffuse large-cell lymphoma. Despite subtle differences in signaling and some biological functions, all fusions appear to be transforming to fibroblasts and hematopoietic cells. Extensive analysis of the leukemogenic potential of NPM-ALK in animal models has further corroborated the importance of NPM-ALK and other ALK rearrangements in the development of ALK-positive ALCL and other diseases.
ALK fusion proteins have also been detected in cell lines and/or primary specimens representing a variety of other tumors including inflammatory myofibroblastic tumor (IMT), neuroectodermal tumors, glioblastomas, melanoma, rhabdomyosarcoma tumors, and esophageal squamous cell carcinomas (see review by Webb T R, Slavish J, et al. Anaplastic lymphoma kinase: role in cancer pathogenesis and small-molecule inhibitor development for therapy. Expert Rev Anticancer Ther. 2009; 9(3): 331-356). Recently, ALK is also implicated in small percent of breast and colorectal cancers (Lin E, Li L, et al. Exon Array Profiling Detects EML4-ALK Fusion in Breast, Colorectal, and Non-Small Cell Lung Cancers. Mol Cancer Res 2009; 7(9):1466-76).
ALK is further implicated in neurological diseases. Evaluation of adult ALK homozygotes, where the ALK kinase domain was deleted, revealed an age-dependent increase in basal hippocampal progenitor proliferation and alterations in behavioral tests consistent with a role for this receptor in the adult brain (Bilsland J G, Wheeldon A, Mead A, et al. Behavioral and neurochemical alterations in mice deficient in anaplastic lymphoma kinase suggest therapeutic potential for psychiatric indications. Neuropsychopharmacology 2008; 33:685-700). These ALK knockout animals displayed an increased struggle time in the tail suspension test and the Porsolt swim test and enhanced performance in a novel object recognition test. Neurochemical analysis demonstrates an increase in basal dopaminergic signalling selectively within the frontal cortex. These results suggest that ALK functions in the adult brain to regulate the function of the frontal cortex and hippocampus and identifies ALK as a new target for psychiatric indications, such as schizophrenia, depression, and substance (cocaine) addiction.
2-amino-pyridines, such as PF-2341066, have been reported as potent inhibitors of the HGF receptor tyrosine kinase (c-Met) and ALK (J. G. Christensen, et al. Abstract LB-271, AACR 2006 meeting; H. Y. Zou et al. Cancer Res 2007; 67: 4408; patent disclosures: WO 2004076412, WO 2006021881, WO 2006021886).

In the phase I trial of PF-02341066 (crizotinib), a dramatic 60% radiographic response rate has been observed in NSCLC patients with EML4-ALK (Kwak E L, Camidge D R, Clark J, et al. Clinical activity observed in a phase I dose escalation trial of an oral c-met and ALK inhibitor, PF-02341066. J Clin Oncol 2009; 27:15s abstract 3509). Subsequent expansion trial confirmed its activity: out of 50 evaluable patients, 64% achieved objective response (ORR) by RECIST, and 90% had disease control (Bang Y, Kwak K L, Shaw D R, et al. Clinical activity of the oral ALK inhibitor, PF-02341066, in ALK-positive patients with non-small cell lung cancer (NSCLC). J Clin Oncol 2010; 28:7s, suppl; abstr 3). It is remarkable that the dramatic response was observed in patients who were refractory to chemotherapies and EGFR inhibitors. Furthermore, most patients had only minor side effects, suggesting that inhibition of ALK is well tolerated. These results firmly validated ALK as a safe and effective target for this subset of NSCLC patients who has no effective alternative systemic treatment options.
Unfortunately, the dramatic response to PF-02341066 is only transient. Most patients develop resistance and the disease progresses after about 6-18 months of treatment. In particular, a significant portion of the patients develop brain metastasis which PF-02341066 is unable to treat.
Previously, we described the substituted pyridazine carboxamide compounds as protein kinase inhibitors (WO 2009/154769). Most of these compounds potently inhibit c-Met and ALK with IC50 of <100 nM. As there is still unmet need in treatment options for kinase mediated diseases such as NSCLC with ALK fusion proteins as described above, herein we further optimized the pyridazine compounds to address the unmet medical needs.